This is a request for an ADAMHA Scientist/Physician Career Development Award, which will enable the candidate to fully focus on research, developing new skills in molecular biology and electrophysiology. The long term objective of the proposed research is to define the molecular mechanisms whereby ethanol and long chain alcohols act at neurotransmitter gated ion channels in the brain. The working hypothesis, based on preliminary data, is that alcohols act at protein sites on ion channels from the nicotinic acetylcholine receptor superfamily. While there is both pharmacologic and physiologic data to implicate neuronal nicotinic receptors in ethanol-induced intoxication and addictive behavior, far more is known about the actions of alcohols at the homologous nicotinic receptors from electroplaque and neuromuscular junction. In these models, long-chain alcohols bind to a hydrophobic inhibitory site within the receptor pore formed by the M2 domains of receptor subunits. Ethanol also interacts with this site, but stabilizes the open channel state. The studies proposed here use site-directed mutagenesis and rapid kinetic electrophysiology techniques to test specific features of the hypothesis in cloned/expressed nicotinic receptors from both muscle and neural tissues.
Specific aim 1 is to define the molecular determinants of alcohol actions on the muscle receptor in order to better formulate models for testing in neuronal receptors. The inhibitory site for alcohols in the receptor pore will be mapped by making mutations that alter the hydrophobicity of specific amino acid side-chains in the M2 domains. To determine why ethanol fails to inhibit channels and how channel stabilization is produced, ethanol's interactions with both the channel site and the receptor's agonist binding sites will be examined.
Specific aim 2 extends these studies into the clinically relevant neuronal nicotinic receptors expressed by either clonal cell lines (PC-12) or by oocytes and cells expressing cloned neuronal subunits. Ethanol and long-chain alcohol actions will be determined in the known functional neuronal receptor subunit mixtures to determine if, as anticipated, the M2 domain structure is a major determinant of drug sensitivity. Specific mechanistic hypotheses based on findings from muscle receptor studies will be tested in neuronal receptors using chimeras or site-directed mutagenesis. This grant will contribute significantly to the candidate's career development by providing time, salary support and supplies necessary for education in neurobiology as well as training in research techniques and strategies to achieve the research goals.
